Characterizing primary transcriptional responses to short term heat shock in Down syndrome

Heat shock stress induces genome-wide changes in transcription regulation, activating a coordinated cellular response to enable survival. We noticed many heat shock genes are up-regulated in blood samples from individuals with trisomy 21. We characterized the immediate transcriptional response to heat shock of two lymphoblastoid cell lines derived from brothers with and without trisomy 21. The trisomy 21 cells displayed a more robust heat shock response after just one hour at 42°C than the matched disomic cells.


Introduction
Lines 9-13.Very long and badly phrased sentence.It is already known (and not "unclear" as state the authors) that the responses from extra chr.21 are not only restricted to the increased expression of genes encoded on chr.21.The following references should be introduced PMID: 34215848, PMID: 36459979, PMID: 36496311, PMID: 27308438, PMID: 25205676 We thank the reviewer for this suggestion.In response we have simplified these sentences and added the additional references requested.They are a great addition to this publication.

Materials and Methods
Lines 43-55 should be moved to the results.Also contains repetitive info e.g. about Nexus biobank (lines 43 and 57).
We have moved the requested lines and removed repetition, as per the reviewer's suggestion.
Line 64.The cells were kept in waterbath at 42 degrees for 1h.So they were kept out of the incubator and lacked CO2 for 1h? Can authors exclude the damaging effect from lack of CO2?If there's any?And separate it from the heat shock?

Ref: PSB 2023
This is an excellent observation from the reviewer.We now include this possibility in the text, specifically see lines 66 and 67.

Results
The figure's labeling does not correspond reality.Please revise.Lines 256-258, line 261 HSF1 levels was not "changes" and was not "not increased" but was "decreased" in certain conditions.This was also shown by Donnelly et al., 2014. Lines 272-274 Repeated sentence.Line 271, 274.You don't have to repeat all the time "1 hour".
We thank the reviewer for his observations and apologize for the lack of clarity on this figure.We have removed repetitive text and revised the labeling.
Lines 287-293 Details more suitable for Materials and Methods section.Line 317.Words are missing in the sentence.Figures 3-5.The two shades of green are very similar and difficult to distinguish on the graphs.Lines 358-360 The title is too long.P values should be indicated on the graphs e.g. of Figure 5 As suggested by the reviewer, we have moved this text to materials and methods.We have also fixed typographical errors and changed the green colors of the graphs to be more distinct.We have also added p-values to figure 5.

Reviewer: 2
In this manuscript, Cardiello et al explore heat shock-related genes from public datasets and EBV-transformed lymphoblastoid cell lines they derive from siblings with and without trisomy 21 (T21).A careful and controlled preliminary analysis is undertaken using clear bioinformatic workflows for molecular assays to identify transcriptional differences between the cell lines where T21 gene dose is a covariate that may be influencing gene expression in the context of heat-shock stress.
Assessing public available RNAseq and proteomic data on a number of tissues from Down syndrome (Human Trisome Project), early gene expression with PROseq and ATACseq for chromatin accessibility, and scRNAseq approaches comparing the lymphoblastoid cell lines, the authors conclude: 1. a number of Heat shock genes are inconsistently upregulated in T21 cohorts compared to disomic controls, including HSF1, a heat shock regulating transcription factor, from publicly available datasets 2. comparison between the lymphblastoid cell lines (T21 and disomic control) show -increased chromatin accessibility at specific heat shock response elements (HSF1, SERPINH1) with heat shock stress with an inferred increased in TF activity using Transcription Factor Enrichment Analysis of ATACseq data, and further (and perhaps difficult to interpret) analysis undertaken for sites enriched for one HSP TF, HSF1 binding by ChIPseq (Fig S3 ) 3. Heat shock response was associated with greater deferentially transcribed genes (up and down) in T21 cell line with enrichment in heat shock associated TFs (HSF1, HSF2, HSF4, HXB2), with PRO-seq suggesting "more robust" nascent RNA transcription in T21 cell line with heat shock.4. Clonal heterogeneity in the cell lines with heat shock using scRNAseq was examined, demonstrating that at least for some certain heat shock responsive genes, changes in transcription appear at some degree to be at a "population" level, rather than dramatically responding outlier clones.
The authors appropriately state that further studies are required to understand the implications of these preliminary, albeit convincing, observations in blood and particularly, immune cells.